Unsaturated derivatives of 7-acylamido-3-cephem-4-carboxylic acid

ABSTRACT

Unsaturated derivatives of 7-acylamido-3-cephem-4-carboxylic acid are disclosed which exhibit antibacterial activity against Gram-positive and Gram-negative microorganisms and can be used to treat infections caused by such microorganisms.

The present invention relates to unsaturated derivatives of7-acylamido-3-cephem-4-carboxylic acid, to a process for theirpreparation and to pharmaceutical and veterinary compositions containingthem. The compounds of the invention have the following formula (I)##STR1## wherein Z is cyano or carbamoyl;

A is trans --CH═CH, cis --CH═CH or --C.tbd.C--;

B is ##STR2## or -S-Het wherein Het is one of the following groups##STR3## wherein R is hydrogen or methyl, or ##STR4## wherein R₁ and R₂are independently selected from the group consisting of hydrogen andmethyl;

X is a free or esterified carboxy group.

Object of the present invention are also the pharmaceutically orveterinarily acceptable salts of the compounds of formula (I) wherein Xis -COOH as well as the matabolites provided with antibacterial activityand the metabolic precursors of the compounds of formula (I).

The pharmaceutically and veterinary acceptable salts of the compounds offormula (I) are those either with inorganic bases, such as, e.g. sodium,potassium, calcium, aluminium hyroxides and alkaline and alkaline-earthcarbonates or bicarbonates, or with organic bases, such as e.g. organicamines, e.g., lysine, triethylamine, procaine, dibenzylamine,N-benzyl-β-phenethylamine, N,N'-dibenzyl-ethylenediamine,dehydroabietilamine, N-ethylpiperidine, diethanolamine,N-methylglucamine, tris-hydroxymethyl-aminomethane and the like.

When X is an esterified carboxy group, it is preferably a group offormula -COOM, wherein M is one of the radicals ##STR5## wherein R₃ ishydrogen or C₁ -C₆ alkyl, Q is --O-- or --NH--, R₄ is an alkyl group(e.g. C₁ -C₆ alkyl) or a basic group, in particular an alkyl (e.g. C₁-C₆ alkyl) or aralkyl (e.g. benzyl) group substituted by at least anamino group, which in turn, may be unsubstituted or substituted, e.g.,R₄ is an alkyl-NH-CH₃, aralkyl-NH-CH₃, ##STR6## --CH₂ --NH₂, R₅ is analkyl group, in particular a C₁ -C₆ alkyl group, e.g., methyl, propyl,isopropyl; an aryl group, in particular phenyl; a cycloalkyl group, inparticular cyclopentyl, cyclohexyl and cycloheptyl; a heteromonocyclicring, e. g., pyridyl; a heterobicyclic ring, e.g., indanyl; an aralkylgroup, e.g., benzyl.

Preferably B is ##STR7## wherein R, R₁ and R₂ are as defined above, andX is free or salified carboxy. Particularly preferred compounds of theinvention are those of formula (I) wherein Z is cyano, A is cis-CH═CH-or -CδC-, B is ##STR8## wherein R, R₁ and R₂ are as defined above and Xis free or salified carboxy; in the latter compounds, A is preferablycis--CH═CH-- and B is preferably ##STR9##

Preferred examples of the compounds of the invention are the following:

(1)7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(2)7-[β-cyano-ethylene(trans-thio-acetamido]-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(3)7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(4)7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(5)-[β-cyano-ethylene(cis)-thio-acetamido]-cephalosporanic acid;

(6)7-[β-carboxamido-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(7)7-[β-carboxamido-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(8) 7-(carboxamido-ethynylene-thio-acetamido)-cephalosporanic acid;

(9)7-carboxamido-ethynylene-thio-acetamido)-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(10)7-(carboxamido-ethynylene-thio-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(11) 7-(cyano-ethynylene-thio-acetamido)-cephalosporanic acid;

(12)7-cyano-ethynylene-thio-acetamido)-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(13)7-(cyano-ethynylene-thio-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(14)7-(cyano-ethynylene-thio-acetamido)-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(15)7-cyano-ethynylene-thio-acetamido)-3-[(5-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(16)7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(5-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(17)7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

(18)7-[β-cyano-ethynylene(trans)-thio-acetamido]-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid,

as well as the pharmaceutically and veterinarily acceptable saltsthereof, in particular the alkaline, preferably sodium and potassium,salts.

The structural formulae of the above-numbered compounds, indicatedaccording to their progressive number, are reported in the followingTable:

                                      TABLE                                       __________________________________________________________________________    Compound                                                                            Z      A          X     B                                               __________________________________________________________________________    1     NC     CHCH(trans)                                                                              COOH                                                                                 ##STR10##                                      2     NC     CHCH(trans)                                                                              COOH                                                                                 ##STR11##                                      3     NC     CHCH(cis)  COOH                                                                                 ##STR12##                                      4     NC     CHCH(cis)  COOH                                                                                 ##STR13##                                      5     NC     CHCH(cis)  COOH  OCOCH.sub.3                                     6     H.sub.2 NCO                                                                          CHCH(trans)                                                                              COOH                                                                                 ##STR14##                                      7     H.sub.2 NCO                                                                          CHCH(cis)  COOH                                                                                 ##STR15##                                      8     H.sub.2 NCO                                                                          CC         COOH  OCOCH.sub.3                                     9     H.sub.2 NCO                                                                          CC         COOH                                                                                 ##STR16##                                      10    H.sub.2 NCO                                                                          CC         COOH                                                                                 ##STR17##                                      11    NC     CC         COOH  OCOCH.sub.3                                     12    NC     CC         COOH                                                                                 ##STR18##                                      13    NC     CC         COOH                                                                                 ##STR19##                                      14    NC     CC         COOH                                                                                 ##STR20##                                      15    NC     CC         COOH                                                                                 ##STR21##                                      16    NC     CHCH(cis)  COOH                                                                                 ##STR22##                                      17    NC     CHCH(cis)  COOH                                                                                 ##STR23##                                      18    NC     CHCH(trans)                                                                              COOH                                                                                 ##STR24##                                      __________________________________________________________________________

the compounds object of the present invention are prepared by a processcomprising:

(a) reacting a compound of formula (II) ##STR25## wherein B and X are asdefined above and E is amino or a group -N═C═Φ, wherein Φ is oxygen orsulphur, or a reactive derivative thereof, with an acid of formula (III)

    z-a-s-ch.sub.2 -cooh                                       (iii)

wherein Z and A are as defined above, or with a reactive derivativethereof; or

(b) reacting a compound of formula (IV) ##STR26## wherein B and X are asdefined above and Y is halogen, or a salt thereof, with a compound offormula (V)

    z-a-sh                                                     (v)

wherein Z and A are as defined above, or with a reactive derivativethereof; or

(c) reacting a compound of formula (VI) ##STR27## wherein B and X are asdefined above, or a reactive derivative thereof, with a compound offormula (VII)

    z-a-y'                                                     (vii)

wherein Z and A are as defined above and Y' is halogen or the residue ofan active ester of an alcohol; or

(d) reacting a compound of formula (VIII) ##STR28## wherein Z, A and Xare as defined above, or a salt thereof, with a compound of formula (IX)

    h-b                                                        (ix)

wherein B is -S-Het, wherein Het is as defined above, or a reactivederivative thereof, so obtaining compounds of formula (I), wherein B is-S-Het, wherein Het is as defined above; and, if desired, converting acompound of formula (I) into a pharmaceutically or veterinarilyacceptable salt and/or, if desired, obtaining a free compound from asalt and/or, if desired, converting a compound of formula (I) or a saltthereof into another compound of formula (I) or a salt thereof.

When in the compounds having the formulae (II), (IV), (VI) and (VIII) Xis a free carboxy group, the carboxy group may be protected, ifnecessary, in a conventional manner before the reaction takes place.

Examples of protecting groups are those usually employed in thesynthesis of peptides, for example, tert-butyl, benzhydryl,p-methoxybenzyl and p-nitrobenzyl. The protecting groups are thenremoved, at the end of the reaction, in a known manner, e.g., by mildacid hydrolysis.

The compounds of formula (I) containing the protecting groups are alsoincluded in the object of the present invention.

A reactive derivative of the compound of formula (II) may be, forexample, an amine salt, a silyl ester or a metal salt when X is carboxy.A reactive derivative of the compound of formula (III) is, for example,an acyl halide, an anhydride or a mixed anhydride, an amide, an azide, areactive ester or a salt, such as, for instance, the salts formed withalkaline or alkaline-earth metals, ammonia or an organic basis.

A reactive derivative of the compounds of formulae (V), (VI) and (IX) ispreferably a salt thereof, for example, an alkaline or alkaline-earthmetal salt.

When Y or Y' are halogen, the halogen is preferably chloride or bromine.Whe Y' is the residue of an active ester of an alcohol, it is preferably-O-mesyl or -O-tosyl.

The reaction between the compound of formula (II) or a reactivederivative thereof and the compound of formula (III) or a reactivederivative thereof may be performed either at room temperature or undercooling, in a suitable solvent, such as, e.g., acetone, dioxane,tetrahydrofuran, acetonitrile, chloroform, methylene chloride,dimethylformamide and, if desired, in the presence of a basis such as,for example, sodium bicarbonate, potassium bicarbonate or atrialkylamine.

When the compound of formula (III) is reacted with the compound offormula (II) wherein E is amino, as a free acid or as a salt, it isdesirable that the reaction be performed in the presence of a condensingagent, such as, for instance, N,N'-dicyclohexylcarbodiimide.

The reaction of the compound of formula (IV), or a salt thereof, withthe compound of formula (IV), or a reactive derivative thereof, forexample a salt, is preferably carried out at temperatures ranging fromabout -30° C. to about + 60° C., preferably at room temperature in thepresence of an inorganic or organic basis, such as, for instance, sodiumor potassium hydroxides, sodium carbonate and triethylamine, in asuitable solvent which may be, for instance, acetone, chloroform,methanol, ethanol, methylene chloride and water. When in the compound offormula (V) the -SH group is not salified, it is preferable to carry outthe reaction in the presence of a basis, such as, for example, analkaline carbonate, an alkaline hydroxide or triethylamine. The reactionbetween the compound of formula (VI), or a reactive derivative thereof,e.g., an alkaline salt, and the compound of formula (VII) may beperformed either in an aqueous medium and in the presence of aninorganic basis or in an organic solvent, preferably in an anhydrousorganic solvent, such as, for example, methylene chloride, chloroform,dioxane, in the presence of an organic basis; the reaction temperaturesrange from about -20° C. to about +50° C.

The reaction between the compound of formula (VIII), or a salt thereof,and the compound of formula (IX), or a reactive derivative thereof, forexample, an alkaline salt, is preferably carried out in an inert organicsolvent, such as, for instance, ethanol, dimethylformamide, dioxane,acetone, methylene-chloride and chloroform; when a salt of the compoundof formula (VIII) is used, e.g., when in the compound of formula (VIII)X is a salified caboxy group, then water or a solvent mixable with wateror a mixture of water and organic solvents such as acetone, ethanol,dioxane and tetrahydrofuran are preferably employed. The rectiontemperatures range from about 5° C. to about 70° C. and the pH fromabout 5 to about 7.5. If necessary, a buffer is used such as, forexample, sodium phosphate or acetate. When in the compound of formula(VIII) X is a salified carboxy group, the salifying agent is preferablyan alkaline or alkaline-earth hydroxide. e

The optional salification of the compound of formula (I) as well as theoptional conversion of a salt into a free compound, may be carried outaccording to conventional methods, i.e., methods already known inorganic chemistry. As stated above, a compound of formula (I) or a saltthereof, may be converted into another compound of formula (I) or a saltthereof; also these optional conversions may be performed by conventonalmethods.

These optional conversions may be e.g., the esterification of a compoundof formula (I), wherein X is carboxy, which may be carried out byreacting the compound of formula (I), wherein the carboxy group is freeor salified, for example in the form of a sodium, potassium, calcium ortriethylammonium salt, with the suitable halide, in an organic solvent,such as acetone, tetrahydrofuran, chloroform, methylene chloride,dimethylformamide, dimethylsulphoxide, or in a mixture of water and anorganic solvent, e.g., dioxane and acetone; the reaction temperaturesrange from about -° C. to about +80° C.

Furthermore a compound of formula (I), wherein X is an esterifiedcarboxy group, may be saponified using, for example, aninorganic acid,such as hydrochloric acid or an inorganic basis, such as sodium orpotassium hydroxide, as is known in organic chemistry.

The compound of formula (II), wherein E is amino, may be prepared, forexample, by reacting 7-amino-cephalosporanic acid or a salt thereof withthe compound of formual (IX), using reaction conditions well known inliterature.

The compound of formula (II), wherein E is -N═C═Φ may be prepared, e.g.,by reacting a compound of formula (II), wherein E is amino, withphosgene or thiophosgene, in the presence of a hydrochloric acidacceptor, using known methods.

The compound of formula (III) may be prepared according to one of thefollowing methods:

(1) by reaction of a compound of formula (VIIa)

    Z-A-Y"                                                     (VIIa)

wherein Z and A are as defined above and Y" is halogen, preferablychlorine or bromine, or tosyl or mesyl, with a compound of formula (X)

    hs-ch.sub.2 -coor                                          (x)

or of a compound of formula (V) with a compound of formula (XI)

    y-ch.sub.2 -coor                                           (xi)

wherein Y is as defined above and R is hydrogen or C₁ -C₆ alkyl,preferably ethyl or tert-butyl; when R is C₁ -C₆ alkyl the reactionproduct is saponified by known methods.

Either the reaction of the compound for formula (VIIa) with the compoundof formula (X) or the reaction of the compound of formula (V) with thecompound of formula (XI) are preferably peformed in water or in anorganic solvent, such as tetrahydrofuran, diethyl ether, benzene, or ina mixture of an organic solvent,e.g., one of those mentioned above, withwater, in the presence of about 2-2.5 equivalents of a basis when R ishydrogen and about 1-1.5 equivalents of basis when R is C₁ -C₆ alkyl andat temperatures ranging from about -10° C. to about +25° C. A suitablebasis is, for example, sodium hydroxide, sodium bicarbonate ortriethylamine.

When in the compounds of formulae (VIIa) and (V) A is cis-CH═CH-, acompound of formula (III) is obtained wherein A is cis-CH═CH-- andviceversa, when in the compounds of formulae (VIIa) and (V) A istrans-CH═-, a compound of formula (III) is obtained wherein A istrans-CH═-;

(2) by reaction of a compound of formula (X) with a compound of formula(XII)

    z-c.tbd.ch                                                 (xii)

so obtaining a compound of formula (III), wherein A is -CH═CH- (cis ortrans).

When the reaction between the compound of formula (X) and the compoundof formula (XII) is performed in a protic solvent, preferably in anaqueous protic solvent, e.g. water or lower aliphatic alcohols, e.g.ethanol in the presence of not more than an equivalent of a basis, e.g.triethylamine, an alkaline bicarbonate, an alkaline hydroxide, and atlow temperatures preferably about 0° C., a compound of formula (III) isobtained wherein A is cis-CH═CH-.

When the same reaction is carried out in the same solvents and in thepresence of the same bases but at temperatures higher than the roomtemperature, or in an excess of the thiolate anion, or with the use ofacid catalysts, e.g. Hcl, a compound of formula (III) is obtainedwherein A is a mixture of cis-CH═CH- trans-CH═CH-.

The separation of the obtained isomers may be carried out by the usualmethods employed in organic chemistry for the separation of geometricisomers such as, for example, fractional crystallization from solventssuch as, for instance, water or lower aliphatic alcohols, e.g. ethanol,or by chromatographic separation.

Furthermore, a compound of formula (III) where Z is cyano may beobtained from the compound of formula (III) wherein Z is carbamoyleither by treatment with a dehydrating agent, such as phosphoruspentachloride, phosphorus oxychloride or triphenylphosphine in anorganic solvent such as a mixture of dimethylformamide and ethyl ether,carbonium tetrachloride, triethylamine and N,N'-dicyclohexylcarbodiimideat room temperature or by heating to about 30-120° C. in an organicsolvent preferably selected from the group consisting ofhexamethylphosphoric triamide and dimethyl sulphoxide.

The compound of formula (IV) may be prepared by reacting the compound offormula (II) with a compound of formula (XI) wherein R is hydrogen orwith a halide or a reactive derivative thereof. The reaction between thecompound of formula (II) and the compound of formula (XI) wherein R ishydrogen, or a halide or a reactive derivative thereof, is preferablycarried out in an aqueous or anhydrous organic solvent such as, e.g.,acetone, dioxane, tetrahydrofuran, acetonitrile, chloroform, methylenechloride, dimethylformamide, in the presence of an organic or inorganicbasis, such as sodium hydroxide or triethylamine, at temperaturesranging from about -10° C. to about +25° C.

For instance, acid halides, anhydrides or mixed anhydrides, azides,amides, reactive esters and salts may be used as reactive derivatives ofthe compound of formula (XI) wherein R is hydrogen.

The compound of formula (VI) may be prepared, for example, by reactingthe compound of formula (II) with a compound of formula (X) wherein R ishydrogen and using reaction conditions analogous to those employed forthe reaction between the compound of formula (II) and the compound offormula (III). The compound of formula (VIII) may be prepared, forexample, by reacting 7-amino-cephalosporanic acid or a salt thereof witha compound of formula (III), using reaction conditions analogous tothose employed for the reaction between the compound of formula (II) andthe compound of formula (III). The compounds of formulae (V), (VII),(VIIa), (IX) as well as the compounds of formulae (X), (XI) and (XII)may be easily prepared by known methods starting from known compoundsor, respectively, they are compounds already known in literature.

The compounds object of the present invention own a high antibacterialactivity either in animals or in humans against both Gram-positive andGram-negative bacteria and are therefore useful in the treatment of theinfections caused by said microorganisms, such as, respiratory tractinfections, for example, bronchitis, bronchopneumonia, pleurisy;hepatobiliary and abdominal infections, for example, cholecystitis,peritonitis; blood and cardiovascular infections, for example,septicemia; urinary tract infections, for example, pyelonephritis,cystitis; obstetrical ad gynecological infections, for instance,cervicitis, endometritis; ear, nose and throat infections, for instance,otitis, sinusitis, parotitis.

The following Table shows the minimal inhibiting concentrations (MIC) inμg/ ml of the compounds of the invention identified by the codes K11457, K 13031, K 13101 and K 13107, against both Gram-positive bacteriaand Gram-negative bacteria in comparison with the known compounds K 9227and K 10299 (which are the most active compounds among those of theBritish Patent specification No. 1.478.055 corresponding to U.S. Patentapplication Ser. No. 485,276 of July 2, 1974), Cefazolin andCefamandole.

                                      TABLE                                       __________________________________________________________________________    (MIC μg/ml)                                                                                  Cefazolin                                                                           Cefamandole                                                                          K 9227                                                                             K 10299                                                                            K 11457                                                                             K 13031                                                                            K 13101                                                                             K                   __________________________________________________________________________                                                              13107               Gram-positive bacteria:                                                       Staphylococcus aureus Smith                                                                     0.05  0.15   0.05 0.075                                                                              0.1   0.1  0.025 0.025               Diplococcus aureus 39/2                                                                         0.6   1.2    0.2  0.4  0.4   0.2  0.14  0.14                Diplococcus pneumoniae ATCC6301                                                                 0.05  0.019  0.4  0.2  0.025 0.012                                                                              0.012 <0.006              Streptococcus β-haemolyticus C203                                                          0.05  0.019  0.025                                                                              0.037                                                                              0.025 <0.012                                                                             0.012 <0.006              Gram-negative bacteria:                                                       Escherichia coli G                                                                              1.6   2.35   3.1  0.8  0.4   0.8  0.14  0.57                Escherichia coli 1507                                                                           1.6   0.8    1.6  0.8  0.4   0.4  0.1   0.57                Klebsiella pneumoniae ATCC 10031                                                                0.8   0.15   0.4  0.2  0.15  0.2  0.1   0.2                 Klebsiella aerogenes 1522E                                                                      1.1   0.4    1.6  0.8  0.2   0.2  0.1   0.28                Enterobacter aerogenes ATCC8308                                                                 1.2   1.6    1.2  0.6  0.4   0.3  0.2   0.4                 Enterobacter cloacae 1321E                                                                      3.1   1.6    3.1  1.6  1.6   0.8  0.56  1.6                 Salmonella typhi Watson                                                                         1.6   0.6    0.8  0.2  0.2   0.2  0.07  0.2                 Shigella sonnei ATCC11060                                                                       1.6   2.35   1.6  1.6  1.6   0.3  0.8   1.6                 Proteus mirabilis ATCC9921                                                                      6.2   1.6    12.5 6.2  3.1   0.8  0.4   0.4                 __________________________________________________________________________     Cefazolin =                                                                   7-(1-(1H)-tetrazolylacetamido)-3-[2-(5-methyl-1,3,4-thiadiazolyl)-thiomet    yl]-3-cephem-4-carboxylic acid;                                                Cefamandole =                                                                 7-D-mandelamido-3-{[(1-methyl-1H-tetrazol-5-yl)-thio]-methyl}-3-cephem-4-    arboxylic acid;                                                                K 9227 =                                                                      7-[(cyanomethyl-thio)-acetamido]-3-[2-(5-methyl-1,3,4-thiadiazolyl)-thiom    thyl]-3-cephem-4-carboxylic acid;                                              k 10299 =                                                                     7-[(cyanomethyl-thio)-acetamido]-3-[5-(1-methyl-1,2,3,4-tetrazolyl)-thiom    thyl]-3-cephem-4-carboxylic acid;                                              K 11457 =                                                                     7-[β-carboxamido-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,    -tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylic acid;                        K 13031 =                                                                     7-[β-carboxamido-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-    etrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylic acid;                          K 13101 =                                                                     7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetraz    l-5-yl)-thiomethyl]-3-cephem-4-carboxylic acid;                                K 13107 =                                                                     7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1,3,4-thiadiazol-2-yl)-    hiomethyl]-3-cephem-4-carboxylic acid.                                    

As is evident from the Table, the compounds of the invention show notonly a high activity against Gram-positive bacteria but surprisinglythey own also a very high activity against Gram-negative bacteria: theyare therefore much more useful than the known compounds of the Table,for the treatment of infections caused by Gram-negative bacteria suchas, for example, urinary tract infections and respiratory tractinfections.

In particular, the Table shows that the compound K 13101 is about 2times more active than Cefazolin against staphylococci, about 4.2 timesmore active than Cefazolin against streptococci (including diplococci)and about 9.6 times more active than Cefazolin against Gram-negativebacteria. Furthermore the compound K 13101 was tested on a series of 60strains of Gram-negative microorganisms comprising Klebsiella,Escherichia coli, Proteus mirabilis and Proteus vulgaris in comparisonwith Cefazolin and the compound K 13101 was always found to be moreactive than Cefazolin. Against the Haemophylus influenzae (Gram-negativebacterium: 7 strains tested), the compound K 13101 was about 6 timesmore active tha Cefazolin. The compounds of the invention may beadministered, either to humans or to animals, in a variety of dosageforms, e.g., orally in the form of tablets, capsules, drops or syrups;rectally in the form of suppositories; parenterally, e.g. intravenouslyor intramuscolarly (as solutions or suspensions), with intravenousadministration being preferred in emergency situation; by inhalation inthe form of aerosols or solutions for nebulizers; intravaginally in theform, e.g. of bougies; or topically in the form of lotions, creams andointments. The pharmaceutical or veterinary compositions containing thecompounds of the invention may be prepared in a conventional way byemploying the conventional carriers and/or diluents used for the othercephalosporins. Conventional carriers or diluents are, for example,water, gelatine, lactose, starches, magnesium stearate, talc, vegetableoils, cellulose and the like. Daily doses in the range of about 1 toabout 100 mg per kg of body weight may be used, in various animalspecies, the exact dose depending on the age, weight and condition ofthe subject to be treated and on the frequency and route ofadministration. A preferred way of administration of the compounds ofthe invention is the parenteral one: in this case the compounds may beadministered, for example to adult humans, in an amount ranging about100 mg to about 200 mg pro dose, preferably about 150 mg pro dose, 1-4times a day, dissolved in a suitable solvent, such as, for examplesterile water or lidocaine hydrochloride solution for intramuscolarinjections, and sterile water, physiological saline solution, dextrosesolution or the conventional intravenous fluids or electrolytes, forintravenous injections.

Furthermore, the compounds of the invention may be used as antibacterialagents in a prophylactic manner, e.g., in cleaning or as surfacedisinfecting compositions, for example, at a concentration of about 0.2to 1% by weight of such compounds admixed with, suspended or dissolvedin conventional inert dry or aqueous carriers for application by washingor spraying. They are also useful as nutritional supplements in animalfeeds.

Assessment of melting points was somewhat difficult in some cases, asthe compounds tend to retain the solvent. In these cases, after theindication of the melting point, the word "dec." (decomposition) wasadded.

The I.R. spectra were determined in a solid phase on a Perkin-Elmer 125spectrophotometer, while the U.V. spectra were usually evaluated in abuffer phosphate solution at pH 7.4 on a Bausch-Lomb apparatus. N.M.R.spectra were determined in DMSO (dimethylsulphoxide) with a VarianHA-100 spectrometer with (CH₃)₄ Si as internal standard. The followingexamples illustrate but do not limit the present invention.

EXAMPLE 1

To a solution containing β-cyano-ethylene(trans)-thio-acetic acid (2.88g) and triethylamine (2.8 g) in anhydrous acetone (120 ml) some drops ofN-methylmorpholine were added. The mixture was cooled at -10° C. andthen pivaloylchloride (2.44 ml) dissolved in anhydrous acetone (30 ml)was added under stirring and then, after stirring for 30 minutes at -10°C., a solution containing7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3cephem-4-carboxylic acid (6.56 g) and triethylamine (2.8 ml) in 50%acetone (240ml), cooled at -20° C., was added after 30 minutes.

The mixture was then stirred at -20° C. for 1 hour and afterwards atroom temperature for 2 hours. The acetone was evaporated under vacuum.The residue was taken up with water, stratified with ethyl acetate andthe pH of the mixture was brought to 2.5 with 40% H₃ PO₄.

After filtration, the ethyl acetate was separated and the organic phasewas washed with water, dried on Na₂ SO₄ and evaporated to small volume.

Ethyl ether was added to give a solid, which was filtered and stirredwith ethyl ether. The solid was filtered again so obtaining 5.4 g (yield60%) of7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid, m.p. 118-120° C. (dec.);

Analysis: Found: C 39.83; H 3.42; N 21.31; S 20.87: C₁₅ H₁₅ N₇ O₄ S₃requires C 39.80; H 3.33; N 21.60; S 21.20;

U.V. (pH 7.4 buffer phosphate): λ_(max) = 267 mμ; E_(1cm) ^(1%) =411;

T.L.C.: R_(f) = 0.55 (CHCl₃ :CH₃ OH:HCOOH= 160:40:20);

I.R. (KBr):

γ (C.tbd.N) conjugated 2200 cm⁻¹ ;

γ (C═O) β-lactam 1775 cm⁻¹ ;

γ (C═O) sec. amide 1670 cm^('1) ;

N.M.R.: ppm (DMSO -d₆);

3.73 (4H, br-s, -S-CH₂ -CO and 2-CH₂);

3.94 (3h, s, CH₃ -N);

4.33 (2h, q, 3-CH₂);

5.11 (1h, d, 6-H);

5.64 (1h, d, NC-CH═);

5.7 (1h, d-d, 7-H) J_(6H-7H) = 4.5 Hz;

7.82 (1H, d, ═CH-S) J_(CH)═CH-trans = 16 Hz;

9.28 (1H, d, --CONH) J_(7H-NH) = 8 Hz.

β-cyano-ethylene(trans)-thio-acetic acid used as starting material wasprepared according to the following methods:

method (A) -- to a solution of 70% thioglycolic acid (2.1 ml) andtriethylamine (5.6 ml) in water (50 ml), cooled at +5° C., a solution oftrans-β-chloroacrylonitrile (1.73 g) in tetrahydrofuran (7 ml) was addeddropwise. The mixture was stirred for 30 minutes at room temperature,acidified with 20% H₂ SO₄. The obtained precipitate was extracted withethyl acetate; the extracts were washed with a saturated solution ofNaCl, dried on Na₂ SO₄, treated with charcoal and evaporated soobtaining an oil which solidifies to giveβ-cyano-ethylene(trans)-thio-acetic acid (2.5 g; yield 88%), m.p.=81-86° C.,

Analysis: Found: C 41.81; H 3.57; N 9.71; S 22.31: C₅ H₅ NO₂ S requiresC 41.94; H 3.52; N 9.78; S 22.39.

I.R. (KBr):

ν(C.tbd.N) conjugated 2220 cm⁻¹ ;

ν(C═O) acid 1720 cm⁻¹ ;

ν(C═C) conjugated 1575 cm⁻¹ ;

ν(C--H): C═C trans 930 cm⁻¹ ;

N.M.R. (DMSO - d₆) : 5.56δ(d,NC--CH═), 7.78 δ(d, ═CH--S);

J_(ch)═ch(trans) = 16 Hz;

method (B) -- to a solution of 70% thioglycolic acid (0.5 ml) and NaHCO₃(0.84 mg) in water (50 ml), β-tosyl-acrylonitrile (1.03 g) was addedunder stirring.

The mixture was stirred for 3 hours at room temperature and filtered.The solution was acidified with 20% H₂ SO₄ and extracted with ethylacetate.

The organic phase was dried on Na₂ SO₄ and evaporated to dryness soobtaining β-cyano-ethylene(trans)-thio-acetic acid (0.65 g; yield 91%).

By purification through the formation of dicyclohexylamine salt aproduct identical to that prepared according to method A was obtained.

β-tosyl-acrylonitrile used as starting material was prepared by reactingsodium salt of p-toluenesulphinic acid and β-chloro-acrylonitrile in amixture of dioxane, water and boric acid according to a known method.

EXAMPLE 2

By proceeding according to Example 1 and reactingβ-cyano-ethylene(trans)-thio-acetic acid with7-amino-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid,7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid, yield 68%, m.p.= 100° C.(dec.) was obtained;

Analysis: Found: C 39.66; H 2.91; N 15.25; S 28.05: C₁₅ H₁₃ N₅ O₄ S₄requires C 39.54; H 2.87; N 15.37; S 28.15

U.V. (pH 7.4 buffer phosphate): λ_(max) = 267 mμ; E_(1cm) ^(1%) = 569.8;

T.L.C.: R_(f) = 0.54 (CHCl₃ :CH₃ OH:HCOOH 160:40:20);

I.R. (KBr): ν (C.tbd.N) conjugated 2215 cm⁻¹ ;

ν(C═O)β-lactam 1775 cm⁻¹ ;

ν(C═O) sec.amide 1675 cm⁻¹.

EXAMPLE 3

To a solution of β-cyano-ethylene(cis)-thio-acetic acid (1.44 g) andtriethylamine (1.4 ml) in anhydrous acetone (80 ml), some drops ofN-methylmorpholine were added. The solution was cooled at 0° C. and thenpivaloylchloride (1.22 ml) dissolved in anhydrous acetone (20 ml) wasadded under stirring. The mixture was stirred at 0° C. for 30 minutes,then a solution containing7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (3.28 g) and triethylamine (1.4 ml) in 50% acetone (160 ml) wasadded.

After the addition the solution was stirred for 1 hour at 0° C. andafterwards for 2 hours at room temperature. The acetone was evaporatedunder vacuum. The residue was taken up with water and washed with ethylacetate. After separation, the aqueous phase was stratified with ethylacetate and brought to pH= 2 with 20% H₂ SO₄.

The residue was filtered off and the organic phase was separated, driedon anhydrous Na₂ SO₄ and evaporated to small volume; the residue wastaken up with ethyl ether so obtaining7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (2.7 g; yield 60%), m.p.= 113°-115° C.(dec.);

Analysis: Found: C 39.78; H 3.43; N 21.40; S 20.78; C₁₅ H₁₅ N₇ O₄ S₃requires C 39.80; H 3.33; N 21.60; S 21.20.

U.V. (pH 7.4 buffer phosphate):λ_(max) =273 mμ; E_(1cm) ¹ % =463;

T.L.C.: R_(f) =0.60 (CHCl₃ :CH₃ OH:HCOOH= 160:40:20);

I.R. (KBr): ν (C═N)conjugated 2210 cm⁻¹ ;

ν(C═O) β-lactam 1775 cm⁻¹ ;

ν(C═O) amide 1680 cm⁻¹ ;

ν(C-N)+ν(N-H) sec.amide 1540 cm⁻¹ ;

N.M.R. ppm (DMSO-d₆);

3.68 (2H, q, 2-CH₂);

3.73 (2h, s,-S-CH₂ -CO);

3.94 (3h, s,CH₃ -N);

4.31 (2h, q, 3-CH₂);

5.10 (1h, d, 6-H);

5.63 (1h, d-d, 7-H);

5.72 (1h, d, NC-CH═);

7.63 (1h, d,=CH-S)J_(CH=CH)(cis) =11 Hz;

9.2 (1H,d,-CONH).

β-cyano-ethylene(cis)-thio-acetic acid used as starting material wasprepared as follows:

To a solution of β-carboxamido-ethylene(cis)-thio-acetic acid (4 g) indimethylformamide/ethyl ether (3:2; 100 ml), cooled at 0° C., phosphoruspentachloride (5.2 g) was added under stirring, maintaining thetemperature between 8° and 10° C. The solution was then stirred for 2hours between 0° and 10° C.

The solution was poured into ice and the ethereal layer was separated;the aqueous layer was extracted four times with ethyl acetate (4× 50ml). The combined extracts were dried on Na₂ SO₄ and then evaporated todryness at a temperature not higher than 40° C., so obtaining ayellowish oil which was dissolved in methanol (10 ml). To the resultingsolution the stoichiometric amount of dicyclohexylamine was added, soobtaining the precipitation of dicyclohexylamine salt ofβ-cyano-ethylene(cis)-thio-acetic acid which, after filtration, wasrepeatedly washed with ethyl ether (m.p.= 180°-183° C.).

The salt was dissolved in water/ethyl acetate (5:7; 120 ml) at 5° C.;the solution was acidified by dropwise addition of 40% H₃ PO₄ (10 ml).The resulting solution was extracted three times with ethyl acetate andthe combined organic extracts were washed with water saturated withNaCl, dried on Na₂ SO₄ and evaporated to dryness under vacuum to obtainβ-cyano-ethylene(cis)-thio-acetic acid (2.76 g; yield 77%; m.p.= 90°-92°C.).

Analysis: Found: C 41.70; H 3.63; N 9.64; S 22.25: C₅ H₅ NO₂ S requires:C 41.94; H 3.52; N 9.78; S 22.39.

I.R. (KBr): ν(C.tbd.N) conjugated 2220 cm⁻¹

ν(C═O) acid 1720 cm⁻¹

N.M.R. (DMSO-d₆):5.4 δ(d, NC-CH═), 7.4 δ(d, ═CH-S) J_(CH)═CH(cis) =10Hz.

EXAMPLE 4

By proceeding according to Example 3 and reactingβ-cyano-ethylene(cis)-thio acetic acid with7-amino-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid,7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid (yield 63%), m.p. 93°-95° C.(dec.) was obtained;

Analysis: Found: C 39.33; H 2.94; N 15.22; S 27.93; C₁₅ H₁₃ N₅ O₄ S₄requires C 39.54; H 2.87; N 15.37; S 28.15.

U.V. (pH 7.4 buffer phosphate):λ_(max) =273 mμ; E_(1cm) ^(1%) =504;

T.L.C. R_(f) =0.56 (CHCl₃ :CH.sub. 3 OH:HCOOH =160:40:20);

I.R. (KBr): ν(C.tbd.N) conjugated 2220 cm⁻¹ ;

ν(C═O)β-lactam 1775 cm⁻¹ ;

ν(C═O)sec. amide 1715 cm⁻¹ ;

ν(C-N)+δ(N-H) sec.amide 1540 cm⁻¹.

EXAMPLE 5

By proceeding according to Example 3 and reactingβ-cyano-ethylene(cis)-thio-acetic acid with 7-amino-cephalosporanicacid, 7-[β-cyano-ethylene(cis)-thio-acetamido]-cephalosporanic acid(yield 70%), m.p.= 132°-134° C.(dec.) was obtained;

Analysis: Found: C 45.15; H 3.93; N 10.33; S 15.99; C₁₅ H₁₅ N₃ O₆ S₂requires C 45.32; H 3.80; N 10.57; S 16.13.

EXAMPLE 6

To a solution of β-carboxamido-ethylene(trans)-thio-acetic acid (0.81 g)in acetonitrile/dimethylformamide (2:1; 60 ml), triethylamine (0.7 ml)and 2 drops of N-methylmorpholine were added. The mixture was cooled at-5° C. and a solution of pivaloylchloride (0.61 ml) in anhydrousacetonitrile (10 ml) was added dropwise uner stirring. After stirringfor 30 minutes at -5° C., a solution of7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (1.64 g) and triethylamine (0.7 ml) in acetonitrile/water (1:1; 70ml) was added, maintaining the temperature at about 0° C. The mixturewas stirred for 1 hour at 0° C., then for 2 hours at room temperatureand evaporated to dryness; the residue was taken up with water andstratified with ethyl acetate and the pH was brought to 2.5 with 40% H₃PO₄. After filtration of the residue and separation of ethyl acetate,the organic phase was washed with water, dried on Na₂ SO₄ and evaporatedto dryness under vacuum. The so obtained raw product was dissolved inmethanol/acetone (1:1; 15 ml) and poured dropwise in ethyl ether (200ml).

After stirring for 2 hours and filtration,7-[β-carboxamido-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid was obtained (1.18 g; yield 50%), m.p.= 146°-150° C.(dec.);

Analysis: Found: C 38.44; H 3.71; N 20.68; S 20.03; C₁₅ H₁₇ N₇ O₅ S₃requires C 38.2; H 3.63; N 20.79; S 20.4.

U.V. (pH 7.4 buffer phosphate): λ_(max) =270 mμ; E_(1cm) ^(1%) =471;

T.L.C.: R_(f) =0.35 (CHCl₃ :CH₃ OH:HCOOH= 160:20:20);

I.R. (KBr): ν(C═O)β-lactam 1780 cm⁻¹ ;

ν(C═O) acid 1670 cm⁻¹ ;

ν(C-N)+δ(N-H) sec.amide 1560 cm⁻¹.β-carboxamido-ethylene(trans)-thio-acetic acid used as starting materialwas prepared as follows: to a solution of 70% thioglycolic acid (2.12ml) in 2N NaOH (20 ml), trans-β-chloro-acrylamide (2.11 g) was addedportionwise at +5° C. The resulting solution was stirred for 3 hours atroom temperature, washed with ethyl acetate; the ethyl acetate wasseparated, the aqueous phase was acidified with 20% H₂ SO₄, so obtainingthe precipitation of β-carboxamido-ethylene(trans)-thio-acetic acid(1.16 g; m.p. 190° C.);

Analysis: Found: C 37.19; H 4.35; N 8.60; S 19.78; C₅ H₇ NO₃ S requiresC 37.25; H 4.37; N 8.69; S 19.89.

I.R. (KBr): ν(N-H) NH₂ group 3420,3290 cm⁻¹ ;

ν(C═O) acid 1690 cm⁻¹ ;

ν(C-H) C═C(trans) 940 cm⁻¹ ;

N.M.R. (DMSO-d.sub. 6): 5.9δ(d, -CO-CH═); 7.63δ(d,═CH-S); J.sub.CH═CH(trans) =15.5 Hz.

EXAMPLE 7

By proceeding according to Example 6 and reactingβ-carboxamido-ethylene(cis)-thio-acetic acid with7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid,7-[β-carboxamido-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (yield 65%), m.p. 150° C.(dec.) was obtained;

Analysis: Found: C 38.35; H 3.72; N 20.43; S 20.17; C₁₅ H₁₇ N₇ O₅ S₃requires C 38.20; H 3.63; N 20.79; S 20.40.

U.V. (pH 7.4 buffer phosphate):λ_(max) =277 mμ; E_(1cm) ^(1%) =409;

T.L.C.: R_(f) = 0.30 (CHCl₃ :CH₃ OH:HCOOH= 160:40:20);

I.R. (KBr):ν(C═O) β-lactam 1775 cm⁻¹ ;

ν(C═O) conjugated amide 1650 cm⁻¹ ;

ν(C-N)+ δ(N-H) sec.amide 1540 cm⁻¹.

β-carboxamido-ethylene(cis)-thio-acetic acid used as starting materialwas prepared as follows: to a solution of propiolamide (6.9 g) in water(20 ml) a solution of 70% thioglycolic acid (10 ml) in 20% NaOH (18.9ml) was added under stirring at about 0° C. The solution was stirredagain for 1 hour at 0° C. and then for 1 hour at room temperature. Atthe end of the stirring the solution was acidifed under stirring withthe stoichiometric amount of 70% perchloric acid. After cooling at about5°-0° C., a solid precipitated which was filtered, taken up with water(45 ml), stirred for 10 minutes, filtered again and dried. 12.6 g of amixture (9:1) of β-carboxamido-ethylene(cis)-thio-acetic acid andβ-carboxamido-ethylene(trans)-thio-acetice acid were so obtained. Thetwo acids have the same solubility in water and therefore by stirringthe mixture with the suitable amount of water it is possible to dissolveall the trans-isomer, leaving still undissolved about 8/9 of thecis-isomer [the purification process can be controlled by thin layerchromatography (acetone/water/acetic acid 180:10:10)]: for instance, bymeans of three subsequent washings (twice with 50 ml of water and thenwith 100 ml of water) 10.9 g of the pure cis-isomer (yield 72%), m.p.=180°-181° C. were obtained;

Analysis: Found: C 37.22; H 4.37; N 8.66; S 20.00; C₅ H₇ NO₃ S requiresC 37.25; H 4.37; N 8.69; S 19.89

I.R. (KBr):γ(N-H) --NH₂ group 3450,3210 cm⁻¹ ;

γ(C═O) acid 1685 cm⁻¹ ;

γ(C═O) amide 1625 cm⁻¹ ;

N.M.R. (DMSO - d₆): 3.43γ(s, --S--CH₂ --); 5.94γ(d, --CO--CH═);

6,97γ(d, ═CH--S); 7.16γ(d, -CONH₂); 12.00γ(br-s OH);

J_(CH)═CH(cis)⁼¹⁰ Hz.

EXAMPLE 8

By using the same method of Example 6 but starting fromcarboxamido-ethynylene-thio-acetic acid instead ofβ-carboxamido-ethylene(trans)-thio-acetic acid,7-(carboxamido-ethynylene-thio-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (yield 47%) was obtained; its structure was confirmed by I.R. andN.M.R. data.

Analogously the following compounds were prepared:

7-(carboxamido-ethynylene-thio-acetamido)-cephalosporanic acid;

7-(carboxamido-ethynylene-thio-acetamido)-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 9

To a solution of cyano-ethynylene-mercapto-acetic acid (1.2 g) inanhydrous acetone (60 ml) and triethylamine (1.24 ml) cooled at -10° c.,isobutylchloroform (1.7 ml) dissolved in anhydrous acetone (16 ml) wasadded under stirring. The stirring was continued for 30 minutes at -10°C., then the mixture was cooled at -30° C.

A solution containing7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (2.8 g) and triethylamine (4 ml) in 50% acetone (120 ml) was thenadded and the resulting mixture was stirred for 1 hour at a temperaturebetween -20° C. and -30° C., subsequently for 1 hour at a temperaturebetween -5° C. to 0° C. and afterwards for 3 hours at room temperature.

The acetone was filtered and evaporated under vacuum; the residue wastaken up with water (200 ml) and extracted with ethyl ether (2× 100 ml).

After separation the aqueous solution was brought to pH 2.5 with 10%hydrochloric acid and extracted with ethyl acetate. The organic phasewas washed with water, dried on Na₂ SO₄, concentrated to small volumeand poured into cyclohexane so obtaining7-(cyano-ethynylene-thio-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid; its structure was confirmed by microanalysis, I.R. and N.M.R.data.

By proceeding analogously, the following compounds were obtained:

7-(cyano-ethynylene-thio-acetamido)-cephalosporanic acid;

7-(cyano-ethynylene-thio-acetamdio)-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

7-(cyano-ethynylene-thio-acetamido)-3-](1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

7-(cyano-ethynylene-thio-acetamido)-3-[(5-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 10

To a solution containing β-cyano-ethylene(cis)-thio-acetic acid (1.44 g)and triethylamine (1.4 ml) in anhydrous acetone (80 ml) some drops ofN-methylmorpholine were added. After cooling at 0° C., a solution ofpivaloylchloride (1.22 ml) in anhydrous acetone (20 ml) was added understirring. The mixture was stirred for 30 minutes at 0° C. and then asolution of7-amino-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid (3.26 g) and triethylamine (1.4 ml) in 50% acetone (160 ml) wasadded. After the addition, the solution was stirred for 1 hour at 0° C.and then for 2 hours at room temperature.

The acetone was evaporated under vacuum, the residue was taken up withwater and washed with ethyl acetate.

After separation of the phases, the aqueous phase was stratified withethyl acetate and the pH brought to 2 with 20% H₂ SO₄.

The residue was filtered off and the organic phase dried on anhydrousNa₂ SO₄ and evaporated to small volume; the residue was taken up withethyl ether. By filtration,7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid (2.5 g; yield 55%), m.p. 125°-130° C.(dec.) was obtained;

Analysis: Found: C 42.77; H 3.96; N 18.27; S 20.87; C₁₆ H₁₆ N₆ O₄ S₃requires C 42.50; H 3.56; N 18.60; S 21.20;

U.V. (pH 7.4 buffer phosphate:λ_(max) =267 mμ; E_(1cm) ^(1%) =458;

T.L.C.: R_(f) =0.32 (CHCl₃ :CH₃ OH:HCOOH=160:40:20);

I.R. (KBr): ν(C.tbd.N) conjugated 2215 cm⁻¹ ;

ν(C═O) β-lactam 1775 cm⁻¹ ;

ν(C═O) sec.amide 1675 cm⁻¹.

Analogously the following compounds were obtained:

7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid, m.p. 127°-131° C.(dec.);

Analysis: Found: C 42.82; H 3.67; N 18.33; S 20.73; C₁₆ H₁₆ N₆ O₄ S₃requires C 42.50; H 3.56; N 18.60; S 21.20.

U.V. (pH 7.4 buffer phosphate): λ_(max) =267 mμ; E_(1cm) ^(1%) =444;

T.L.C.: R_(f) =0.28 (CHCl₃ :CH₃ OH:HCOOH=160:40:20);

I.R. (KBr): ν(C.tbd.N) conjugated 2215 cm⁻¹ ;

ν(C═O) β-lactam 1775 cm⁻¹ ;

ν(C═O) sec.amide 1675 cm⁻¹ ;

7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(5-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid;

Analysis: Found: C 42:63; H 3.73; N 18.40; S 20.91; C₁₆ H₁₆ N₆ O₄ S₃requires C 42.50; H 3.56; N 18.60; S 21.20.

EXAMPLE 11

To a solution of7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (3.28 g) and NaHCO₃ (2g) in 50% aqueous acetone (60 ml), cooled at0° C., a solution of 2.18 g of β-cyano-ethylene(trans)-thioacetic acidchloride (obtained from the acid by reaction with oxalyl chloride indimethylformamide at 0° C.) in acetone (30 ml) was added under stirring.The mixture was stirred for 20 minutes at a temperature between 0° C.and 5° C. The acetone was evaporated, ethyl acetate was added to theresulting aqueous solution which was then acidified with 8% hydrochloricacid to pH 2. The organic phase was washed with water, dried andevaporated under vacuum. The residue was treated with ethyl ether andfiltered so obtaining7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4,-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (2.3 g), m.p. 118°-120° C.(dec.); microanalysis, U.V., T.L.C., I.R.and N.M.R. data of this compound were identical to those alreadyreported in Example 1. By proceeding analogously, also the othercompounds of the invention mentioned in the preceding Examples wereprepared.

EXAMPLE 12

To a solution containing7-(bromo-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (4.45 g) in CH₂ Cl₂ (50 ml), triethylamine (2.8 ml) was added.

After stirring for 30 minutes at room temperature,trans-1-cyano-2-mercaptoethylene (0.85 g) was added and the mixture wasstirred for 6 hours at room temperature.

The obtained precipitate was filtered off; the organic solution waswashed with water, dried and evaporated to dryness under vacuum. Theresidue was taken up with ethyl ether so obtaining7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (3.8 g), m.p. 118°-120° C.(dec.); microanalysis, U.V., T.L.C., I.R.and N.M.R. data of this compound were identical to those alreadyreported in Example 1.

By proceeding analogously, also the other compounds of the inventionmentioned in the preceding Examples were obtained.

7-(bromo-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid used as starting material was prepared by reacting, using a knownmethod, bromoacetylbromide with7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 13

To a solution containing7-(mercapto-acetamido)-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (4.22 g) in CH₂ Cl₂ (70 ml), triethylamine (2.8 ml) was added.

The mixture was stirred for 30 minutes at room temperature and thentrans-β-chloroacrylonitrile (0.87 g) dissolved in CH₂ Cl₂ (15 ml) wasadded.

After stirring for 4 hours at room temperature, the obtained precipitatewas filtered off and the organic solution was washed with water, driedand evaporated to dryness under vacuum; the residue was taken up withethyl ether, so obtaining7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (3.6 g), m.p. 118°-120° C. (dec.).; microanalysis, U.V., T.L.C.,I.R. and N.M.R. data of this compound were identical to those alreadyreported in Example 1. By proceeding analogously, also the othercompounds of the invention mentioned in the preceding Examples wereobtained.

EXAMPLE 14

To a solution of sodium salt of7-[β-cyano-ethylene(cis)-thio-acetamido]-cephalosporanic acid (4.19 g)in acetone (40 ml) and pH 7 buffer phosphate (200 ml),5-mercapto-1-methyl-1,2,3,4-tetrazole (1.3 g) and NaHCO₃ (1.84 g) wereadded and the mixture was stirred for 6 hours at 60° C.

After cooling, stratification with ethyl acetate and acidification with10% hydrochloric acid, the pH was brought to 2. The two phases systemwas filtered and the organic phase separated. The aqueous phase wasbrought to pH= 4.5 with 10% ammonium hydrate and extracted with ethylacetate; the extracts were washed with water, dried and evaporated todryness, so obtaining7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (yield 65%), m.p. 113°-115° C. (dec.); microanalysis, U.V., T.L.C.,I.R. and N.M.R. data of this compound were identical to those alreadyreported in Example 3.

By proceeding analogously, also the other compounds of the inventionmentioned in the preceding Examples were obtained.

EXAMPLE 15

To a solution of β-cyano-ethylene(cis)-thio-acetic acid (1.44 g) inanhydrous tetrahydrofuran (50 ml), dicyclohexylcarbodiimide (2.1 g) wasadded and the mixture was stirred for 30 minutes at room temperature. Tothis mixture, a solution of7-amino-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (3.28 g) and NaHCO₃ (0.84 g) in tetrahydrofuran/water (1:1; 60 ml)was added. After stirring for 3 hours at room temperature thetetrahydrofuran was evaporated under vacuum; the residue was taken upwith water and the dicyclohexyl-urea was filtered off.

The filtrate was stratified with ethyl acetate, acidified with 20% H₂SO₄ to pH 2.5; the organic layer was separated, washed with water,evaporated to small volume and then ethyl ether was added to give asolid which was filtered and then stirred with ethyl ether so obtaining7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid, m.p. 113°-115° C. (dec.); microanalysis, U.V., T.L.C.,I.R. andN.M.R. data were identical to those already reported in Example 3.

By proceeding analogously, also the other compounds of the inventionmentioned in the preceding Examples were obtained.

EXAMPLE 16

To an aqueous suspension of7--[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (4.53 g) in water (80 ml), the stoichiometric amount of NaHCO₃ wasadded, so obtaining the complete solution of the compound. This solutionwas then lyophilized so obtaining sodium salt of7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 17

To a solution of7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (1.13 g) in ethyl acetate (30 ml), the stoichiometric amount of a30% solution of sodium 2-ethyl-hexanoate in isopropyl alcohol was added.

After stirring for 30 minutes at room temperature, the mixture wasdiluted with petroleum ether and the obtained precipitate was filteredto give sodium salt of7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 18

To a suspension of sodium salt of7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid (2.38 g) in acetone (40 ml), a 10% solution of sodium iodide inwater (0.4 ml) and chloromethylpivalate (0.72 ml) was added.

The suspension was heated for 3 hours under reflux and after coolingat5° C., the solid was filtered off and the resulting solution evaporatedunder vacuum. The oily residue was dissolved in ethyl acetate (50 ml),the resulting solution washed with a 5% solution of NaHCO₃ in water andthen with water, dried and evaporated to dryness so obtaining thepivaloyloxymethyl ester of7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 19

An injectable pharmaceutical composition was performed by dissolving100-500 mg of sodium7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylatein sterile water or steril normal saline solution (1-2 ml).

We claim:
 1. A compound of formula (I) ##STR29## wherein Z is cyano orcarbamoyl;A is trans --CH═CH or cis --CH═CH; B is -S-Het wherein Het isone of the following groups ##STR30## wherein R is hydrogen or methyl,or ##STR31## wherein R₁ and R₂ are independently selected from the goupconsisting of hydrogen and methyl; X is a free or esterified carboxygroup, and the pharmaceutically or veterinarily acceptable salts of thecompound of formula (I) wherein X is a free carboxy group.
 2. Compoundof claim 1, wherein X is a free carboxy group or a pharmaceuticallyacceptable salt thereof.
 3. The compound of claim 1, wherein Z is cyano,A is cis--CH═CH--, B is ##STR32## wherein R, R₁ and R₂ are as defined inclaim 1 and X is free or salified carboxy.
 4. The compound of claim 3,wherein A is cis--CH═CH-- and B is ##STR33## 5.7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 6.7-[β-cyano-ethylene(trans)-thio-acetamido]-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 7.7-[β-cyano-ethylene(cis)-thio-acetamido]-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 8.7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1,3,4-thiadiazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 9.7-[β-carboxyamido-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 10.7-[β-carboxamido-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,2,3,4-tetrazol-5-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 11.7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(5-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 12.7-[β-cyano-ethylene(cis)-thio-acetamido]-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid. 13.7-[β-cyano-ethylene(trans)-thio-acetamido]-3-[(1-methyl-1,3,4-triazol-2-yl)-thiomethyl]-3-cephem-4-carboxylicacid,
 14. A pharmaceutically or veterinarily acceptable salt of each ofthe compounds of claims 5 to 8, 9, 10 and 11 to
 13. 15. The salt ofclaim 14, wherein said salt is an alkaline salt.
 16. The salt of claim15 wherein said salt is the sodium salt.
 17. The salt of claim 15,wherein said salt is the potassium salt.
 18. Compound of claim 1,wherein Het is ##STR34## wherein R is defined in claim
 1. 19. A compoundof the formula (I) ##STR35## wherein Z is cyano or carbamoyl;A iscis--CH═CH--; ##STR36## X is carboxy, and the pharmaceutically orveterinarily acceptable salts thereof.
 20. A pharmaceutical orveterinary composition suitable for the treatment of Gram-positive andGram-negative microorganisms, said composition comprising atherapeutically effective amount of a compound according to claim 1, anda pharmaceutically or veterinarily acceptable carrier or diluent.
 21. Amethod of treating a patient suffering from conditions caused byGram-positive and Gram-negative microorganisms, said method comprisingadministering to said patient a therapeutically effective amount of acompound of claim
 1. 22. A method according to claim 21, wherein saidcompound is administered orally or parenterally.